(Protein Degradation and Cholesterol Regulation) HMG-CoA reductase (HMGR) is a key enzyme of the sterol pathway that produces a variety of essential molecules. HMGR is an integral membrane ER protein and is subject to regulated destruction mediated by ER-associated degradation (ERAD). Our initial discovery that HMGR regulated degradation is conserved in yeast has allowed us uses the uniquely facile approaches to unravel the underlying mechanisms of HMGR ERAD and its regulation by the sterol pathway. The yeast HMGR isozyme Hmg2 undergoes ubiquitin-mediated ER degradation by the HRD quality control pathway. HRD- dependent Hmg2 degradation is controlled by levels of the sterol pathway molecule farnesyl pyrophosphate (FPP): elevated FPP leads to increased entry into the HRD degradation pathway. We have made substantial progress towards understanding how the HRD machinery recognizes Hmg2 and other substrates, and how the sterol pathway controls entry of Hmg2, a normal protein, into the HRD quality control pathway. In the past funding period we have found remarkable similarities between the yeast and mammalian systems, including the nature of the signals, the use of ERAD as the degradative mediator, the involvement of conserved motifs, and the participation of INSIGs to impart sterol control. Using uniquely available tools we developed for these studies we plan to push forward our parallel paths of study on HRD mechanisms and sterol pathway signaling. We will 1) Continue our study of the HRD E3 ligase complex, focusing on understanding the mechanism of misfolded membrane protein detection, and HRD complex regulation, 2) Analyze the mechanism of Hmg2 retrotranslocation from the ER membrane using a new in vitro assay developed by our group, in conjunction with genetic and proteomic approaches to discern the participating molecules in this still-mysterious process, 3) Study the features of Hmg2 allowing regulation by sterol pathway signals - with a particular emphasis on the highly conserved sterol sensing domain (SSD) of Hmg2 and the INSIG Nsg1 that we have discovered imparts sterol-mediated control on Hmg2 degradation, and 4) Discover the nature and action of the sterol pathway signals that control Hmg2 ERAD testing the hypothesis that GGPP is the actual FPP-derived degradation signal and the model that GGPP causes Hmg2 to undergo regulated misfolding to trigger HRD pathway entry. These studies provides the double benefit of revealing the tactics employed by cells to measure and modify sterol synthesis, and the nature of a protein quality control pathway of great basic and biomedical interest.